Auxin sensing is a property of an unstructured domain in the Auxin Response Factor ETTIN of Arabidopsis thaliana

Abstract

The plant hormone auxin regulates numerous aspects of the plant life cycle. Auxin signalling is mediated by auxin response factors (ARFs) that dimerise with modulating Aux/IAA repressors. ARF3 (ETTIN or ETT) is atypical as it does not interact with Aux/IAA repressors. It is proposed to be a non-canonical auxin sensor, regulating diverse functions essential for development. This sensing ability relies on a unique C-terminal ETT specific domain (ES domain). Alignments of ETT orthologues across the angiosperm phylum revealed that the length and sequence identities of ES domains are poorly conserved. Computational predictors suggested the ES domains to be intrinsically disordered, explaining their tolerance of insertions, deletions and mutations during evolution. Nevertheless, five highly conserved short linear motifs were identified suggesting functional significance. High-throughput library screening identified an almost full-length soluble ES domain that did not bind auxin directly, but exhibited a dose-dependent response in a yeast two-hybrid system against the Arabidopsis INDEHISCENT (IND) transcription factor. Circular dichroism confirmed the domain was disordered. The identification and purification of this domain opens the way to the future characterisation of the ETT auxin-sensing mechanism in planta and an improved understanding of auxin-mediated regulation.

Figure 1

Analyses of ETT domains, order-disorder prediction and secondary structure prediction. (A) Protein sequence alignment by SIM between ETT/ARF3 and its closest homologue ARF4. Different colours indicate percentage similarity. Below, schematic representation of the ETT showing limit of DNA binding domain (cyan) as determined by alignment with ARF4 and the following ETT-Specific (ES) domain (dark green). (B) Y2H assay between the ETT and IND, an A. thaliana transcription factor previously identified as ETT interactor, and ETT-ES domain and IND. Interaction is observed on the selective plate -W-L-H-A. Yeast growth is inhibited by IAA in the media (lower panel). (C) A disorder prediction and putative protein binding regions for full-length ETT by PrDos and DISOPRED3 servers. (D) Secondary structure prediction by SOPMA.

Figure 2

Four motifs are conserved in the ES domain across angiosperms. (A,B) Schematic representation of the conserved motifs identified in the ES domain (A) and their sequence and possible function (B). (C,D) Alignment of ES domain from different angiosperm species (C) and their phylogenetic tree (D). Motif colour code: red, NLS; yellow, Motif 1A and 1B; blue, Ser patch; purple, Motif 2. (E) Alignment between ETT/ARF3 and ARF4 highlighting conservation of NLS, Motif 1A and 1B and Motif 2 between the two proteins.

Figure 3

Screening for expression and solubility from a ETT random truncation library. (A) Schematic representation of the three ETT C termini used in N-terminal truncation experiments: aa 1–608, 1–602 and 1–594. (B) Schematic representation of the strategy for identifying the soluble ES domain of ETT. From a high throughput screen of 15,975 clones, 12 clones were selected for 1 litre scale-up. (C) Summary of sequenced ETT constructs yielding purifiable fragments after 4 mL scale-up. Construct boundaries are shown with the predicted molecular weights including 5 kDa from the hexahistidine and biotin tags. (D) Fluorescent streptavidin blot against the C-terminal BAP for protein fragments summarised in (C). Uncropped gel shown in Supp. Fig. 4A,B. (E) CD analysis of the ETT_388-594 construct showing characteristic spectrum of an unstructured protein.

Figure 4

ETT 388–594 is the auxin-responsive domain. (A) Schematic representation of ETT variants and their behaviour in Y2H assay in combination with IND and in the absence and presence of auxin at different concentrations. Clones EP1, 2 and 3 were isolated from a random mutagenesis analysis of the ES domain; clones called ES_SP, referring to the Ser patch, are serine to threonine mutations introduced by site-specific mutagenesis. (B–E) Mapping and Scanning electron microscopy images of gynoecium of annotated A. thaliana wild-type (B) and ett mutant: arf3-1 (D), arf3-2 (E) and ett-3 (F). (G–J) Mapping and optical microscopy images of gynoecium of Brassica rapa wild type (G) and ett mutant: BraA.ett.a Stop220 (I) and BraA.ett.a Gln480Stop (J). Abbreviations: rp, replum; sg, stigma; st, style, va, valves. Scale bars: 100 μm (B,D,E), 1 mm (G,I,J).